KRAS mutations as essential promoters of lymphangiogenesis via extracellular vesicles in pancreatic cancer

Kirsten rat sarcoma virus (KRAS) gene mutations are present in more than 90% of pancreatic ductal adenocarcinomas (PDACs). KRASG12D is the most frequent alteration, promoting preneoplastic lesions and associating with a more aggressive phenotype. These tumors possess increased intratumoral lymphatic networks and frequent lymph node (LN) metastases. In this issue of the JCI, Luo, Li, et al. explored the relationship between the presence of the KRASG12D mutation and lymphangiogenesis in PDAC. The authors used in vitro and in vivo models and an elegant mechanistic approach to describe an alternative pathway for lymphangiogenesis promotion. KRASG12D induced SUMOylation of heterogenous nuclear ribonucleoprotein A1 (hnRNPA1) via SAE1 and SUMO2 activation. SUMOylated hnRNPA1 was loaded into extracellular vesicles (EVs) and internalized by human endothelial lymphatic cells (HLEC). Further, SUMOylated hnRNPA1 promoted lymphangiogenesis and LN metastasis by stabilizing prospero homeodomain protein 1 (PROX1) mRNA. These data provide mechanistic insight into cancer lymphangiogenesis with the potential for developing biomarkers and RAS pathway therapeutics.

Pancreatic cancer is the deadliest gastrointestinal malignancy, as the fourth leading cause of cancer-related death and with one of the lowest 5-year survival rates, at only 11% (1). Pancreatic cancer is most often detected in advanced stages -when few therapeutic options are available -due to the lack of screening methods and paucity of symptoms in early phases. The main histologic type is pancreatic ductal adenocarcinoma (PDAC), which carries KRAS gene mutations as molecular signatures in more than 90% of cases (2). As a driver gene in pancreatic cancer, KRAS alteration promotes the development of epithelial dysplasia, pancreatic intraepithelial neoplasias (PanINs), and pancreatic mucinous neoplasms (IPMNs) (3). Until recently, KRAS mutations were known as undruggable targets due to the structural difficulty of designing specific inhibitors (4). In pancreatic cancer, KRAS G12D is the most common KRAS mutation, inducing an aggressive phenotype via the activation of MAPK, PI3K, and Ras-like GEF (RalGEF) pathways (3, 5). Pancreatic tumors harboring the KRAS G12D mutation show a particular disposition -with tumor cells surrounding lymphatic vessels, lymphatic vessel remodeling, and increased lymphangiogenesis -which facilitates intratumoral lymphatic vessel invasion and LN spreading (6). Consequently, two-thirds of patients with PDAC present with LN metastases at the time of diagnosis (7).

KRAS G12D -driven SUMOylation of hnRNPA1
A more in-depth understanding of the genomic landscape of cancer using translational approaches can provide insights into the genetic alterations and unravel oncogenic pathways and possible therapeutic targets (8). In this issue of the JCI, Luo, Li, et al. carefully investigated the molecular mechanism behind KRAS G12D -induced lymphangiogenesis in pancreatic cancer. The authors used a large spectrum of in vitro and in vivo models to analyze intercellular communication between malignant cells and human endothelial lymphatic cells (HLEC) by characterizing the EVs and intracellular pathways (9). Based on the group's previous results, which show that lymphangiogenesis is promoted by cancer-related genes interacting with RNA binding proteins, the authors investigated the role of heterogenous nuclear ribonucleoprotein A1 (hnRN-PA1) in promoting lymphangiogenesis in PDAC. Notably, hnRNPA1 is overexpressed in PDAC, with expression levels correlating with the KRAS G12D mutation (10).
Analysis of the hnRNPA1 expression in serum samples from patients with PDAC KRAS G12D mutations collected from two independent clinical centers showed that elevated hnRNPA1 was associated with reduced overall and disease-free survival. In KRAS G12D PDAC, hnRNPA1 is overexpressed in EVs and LN metastases, suggesting that hnRNPA1 promotes LN metastasis via intercellular shuttling. The influence of hnRNPA1 on promoting PDAC metastasis was highlighted by Luo, Li, et al. using a popliteal lymphatic metastasis mice model (9). EVs high in hnRNPA1 acted directly Kirsten rat sarcoma virus (KRAS) gene mutations are present in more than 90% of pancreatic ductal adenocarcinomas (PDACs). KRAS G12D is the most frequent alteration, promoting preneoplastic lesions and associating with a more aggressive phenotype. These tumors possess increased intratumoral lymphatic networks and frequent lymph node (LN) metastases. In this issue of the JCI, Luo, Li, et al. explored the relationship between the presence of the KRAS G12D mutation and lymphangiogenesis in PDAC. The authors used in vitro and in vivo models and an elegant mechanistic approach to describe an alternative pathway for lymphangiogenesis promotion. KRAS G12D induced SUMOylation of heterogenous nuclear ribonucleoprotein A1 (hnRNPA1) via SAE1 and SUMO2 activation. SUMOylated hnRNPA1 was loaded into extracellular vesicles (EVs) and internalized by human endothelial lymphatic cells (HLEC). Further, SUMOylated hnRNPA1 promoted lymphangiogenesis and LN metastasis by stabilizing prospero homeodomain protein 1 (PROX1) mRNA. These data provide mechanistic insight into cancer lymphangiogenesis with the potential for developing biomarkers and RAS pathway therapeutics. J Clin Invest. 2022;132(14):e161454 https://doi.org/10.1172/JCI161454 small non-coding microRNAs to enhance their loading into EVs (13). Going deeper into understanding the hnRNPA1 modification by SUMOylation, Luo, Li, et al. showed that SUMOylation was triggered by the E1 SUMO-activating enzyme (SAE1), which depended on the activation of KRAS G12D mutation-induced KRAS/RAF signaling. Experimental upregulation of SAE1 induced hnRNPA1 SUMOylation at lysine residue 113 (K113) (9). Further, SUMOylated hnRN-PA1 K113 interacted directly with the tumor susceptibility gene 101 protein (TSG101), which increased the loading of SUMOylated hnRNPA1 into EVs (9). TSG101 is a key element for the endosomal sorting complex responsible for transport (ESCRT) mechanism. This component triggers EV synthesis and the development of intracellular vesicles that form multivesicular bodies. Subsequent fusion with the plasma membrane releases the vesicular cargo into the extracellular space (14). The interaction between TSG101 and SUMOylated hnRNPA1 K113 was essential for effective loading of the hnRNPA1 into PDAC EVs and lymphangiogenesis promotion (Figure 1) (9).

From mutated KRAS to lymphangiogenesis
Luo, Li, et al. showed that HLECs from the local tumor microenvironment internalized EVs rich in SUMOylated hnRNPA1 that were released from PDAC cells by exocytosis ( Figure 1) (9). Cytosolic PDAC-derived SUMOylated hnRNPA1 present in HLEC cells promoted tube formation and migration. The cytosolic hnRNPA1 promoted lymphangiogenesis in HLEC by upregulating PROX1 expression. PROX1 is a master regulator of lymphatic system development, necessary for lymphangiogenesis and essential for endothelial cell differentiation toward an HLEC phenotype (15). Luo, Li, et al. showed that hnRNPA1 derived from PDAC EVs -once endocytosed and released into the cytosol -interacts with an AU-rich region in the PROX1 3′-untranslated region, increasing PROX1 expression by stabilizing the mRNA to increase its half-life. The hnRNPA1-induced lymphangiogenesis in HLEC cells takes place independent of VEGF-C, as it was shown that anti-VEGF-C antibodies did not interfere with the hnRNPA1 effect on PROX1 (9). The effect of KRAS G12D PDAC EVs on LN metastasis and lymphangiogenesis was form, suggesting the presence of additional structural post-translational modifications (PTMs). The authors screened for PTMs involved in hnRNPA1 loading into EVs and found that a SUMOylation modifier, SUMO2, was directly bound to hnRNPA1, enhancing its packaging in PDAC EVs (9).
SUMOylation, small ubiquitin-like modifier binding, is an essential PTM mechanism that mediates protein stability and subcellular localization (11). SUMOylation is an essential process for packaging protein cargos into EVs. It was previously shown that hnRNPA1 is prone to SUMOylation (12) and that SUMOylated hnRNPA1 binds with on lymphatic endothelial cells to increase lymphangiogenesis and LN metastasis. Interestingly, these effects on lymphangiogenesis were observed only using the PANC-1 cell line, which harbors the KRAS G12D mutation (9). These results suggest that the KRAS G12D protein has an active role in upregulating hnRNPA1 protein expression.
Additional investigation into the molecular mechanism by which KRAS G12D upregulates hnRNPA1 revealed a structural difference between cytosolic hnRNPA1 and the hnRNPA1 loaded in EVs. When isolated from PDAC EVs, hnRNPA1 had a higher molecular weight than the intracellular KRAS inhibitors may provide an elegant blocking strategy. In 2021, two molecules, sorafenib and adagrasib, were FDA approved for tumors harboring the KRAS G12C alteration, opening the avenue for KRAStargeted therapies (19,20). Also, other KRAS inhibitors undergoing preclinical investigations show promising results, moving the field toward early-phase clinical trials. Additional strategies could analyze approaches that combine classic anti-VEGF molecules with those targeting this VEGF-independent lymphangiogenesis pathway to better control KRAS-mutated pancreatic cancers. Targeting lymphangiogenesis has been a challenge in recent years and there are currently no FDA-approved molecules that specifically inhibit lymphangiogenesis (21). Previous approaches -focused on lymphangiogenesis-pathway regulators, such as VEGF-C and VEGFR-3 -had mixed results for both the in vitro and in vivo models (22). Therefore, a possible combination of VEGF-C, KRAS and hnRNPA1 inhibitors could enhance an inhibitory effect on the tumor-induced lymphangiogenesis.
In conclusion, Luo, Li, et al. unraveled a molecular mechanism behind tumorinduced lymph vessel development and early-LN metastasis in KRAS G12D PDAC. The authors identified KRAS G12D protein as an essential factor for VEGF-C-independent induction of lymphangiogenesis. Mechanistically, KRAS G12D had a role in activating SAE1-dependent SUMOylation of hnRN-PA1 with subsequent EV loading, which further induced PROX1 activation in HLEC (9) (Figure 1). This exciting basic research has substantial translational implications.